Worldwide maize (corn) is in large demand as a source of calories for animal and human consumption. The availability of maize can be impacted by nature through weather disasters or by insects or by plant disease. Nature's impact is especially strong because the majority of the maize is grown in limited areas. Thus, insects and plant disease are often transmitted from field to field and cover large corn growing regions. Corn production can be significantly decreased by maize diseases such as Head Smut, CLN, Gray Leaf Spot, Rust, and Maize Chlorotic Dwarf Virus (MCDV). Because of the impact that pests and disease can have on the production of corn, there has understandably been great public interest in developing plants that are disease resistant or pest resistant. Correspondingly, there has been extensive research in breeding corn lines that have these types of resistance.
Occasionally, disease resistant maize is developed by traditional breeding methods; unfortunately however, there is usually a reduction in desirable agronomic traits of the maize plant when disease resistance is introduced. This trade off between desirable agronomic traits in a corn plant and the disease resistances are primarily due to the traditional method by which disease resistance is transferred into the elite inbred. Plants that have disease resistance frequently are unadapted germplasm. Adapted germplasm is germplasm which is specifically bred and selected for use in a commercial environment whereas unadapted germplasm is usually not bred for commercial use. Often unadapted germplasm contains the desired resistance and therefore unadapted germplasm is employed to introduce the desired disease resistance into adapted commercial germplasm. A large portion of chromosomal material from the genotype of the unadapted germplasm is crossed or otherwise introduced into the adapted germplasm. Because the portion of the unadapted germplasm which contains disease resistance is not identified nor located, it cannot be readily tracked when introduced into the adapted germplasm. Therefore, when carrying the disease resistance portion of the genotype from the unadapted germplasm into the adapted germplasm, a variety of the unadapted germplasm's less desirable agronomic traits are often retained and expressed in the resultant resistant plant.
The CLN disease in maize is a disease complex that consists of not just one virus, but at least two viruses to infect the plant. The CLN disease complex usually requires a combination of MDMV.sub.B and maize chlorotic mottle virus (MCMV) to infect a single plant. Traditional methods of plant breeding have been employed in an attempt to transfer resistance to CLN or MDMV.sub.B into commercially viable germplasm for decades. The result has been an occasional inbred line which shows some tolerance; however, the ability to produce a truly resistant plant with genetically desirable traits requires identification of the location of the resistant chromosomal material. Most corn lines considered to be tolerant to CLN tend to lack desirable agronomic traits such as low grain moisture at harvest, high yields per acre, and low percentage of stalk or root lodging.
MDMV has a variety of strains. There is MDMV strain A, MDMV.sub.B along with other types of strains which have been identified. There has been an ongoing debate on how and where in the potyvirus groups MDMV.sub.B should be classified because the status of Sugar Cane Mosaic Virus (SCMV) strains have not been clearly identified. Within the strains, there appear to be four discernible potyvirus groups: Johnsongrass Mosaic Virus (JGMV), Maize Dwarf Mosaic Virus (MDMV), Sorghum Mosaic Virus (SrMV), and SCMV. Through protein profiles of seventeen strains, MDMV.sub.B has been identified as being classified among the Sugar Cane Mosaic Virus (SCMV) group. Additionally other strains of SCMV include: Sugar Cane Mosaic Virus strain A, Sugar Cane Mosaic Virus strain BC (SCMV.sub.BC), Sugar Cane Mosaic Virus strain D, Sugar Cane Mosaic Virus strain Isis, and Sugar Cane Mosaic virus-SC. There also appears to be a subset of profiles within this group including Sugar Cane Mosaic Virus strain BC and MDMV.sub.B. These two are more closely linked by profile than are the other four viruses. This subset may be associated with the fact that neither MDMV.sub.B nor SCMV.sub.BC tend to infect sugar cane.
MDMV strains are distributed worldwide and are important in their affect both on the growth and yield of dent corn and sweet corn. Two strains which are detrimental to the United States corn industry are MDMV.sub.A which is found usually in the eastern United States, and MDMV.sub.B, which occurs in the Midwest.
Irrespective of how MDMV.sub.B is taxonomically identified, it is an essential component of CLN. Thus resistance to MDMV.sub.B in a plant usually results in resistance to CLN. MDMV.sub.B is transmitted most often by an insect vector, however, it can be transmitted through mechanical means directly from infected plant to noninfected plant. MCMV.sub.B is frequently vectored by greenbugs and corn leaf aphids. Other vectors of MDMV.sub.B include Rhopalosiphum maidis, R. padi, Myzus persicae, and Schizaphis graminum. MCMV, which is another component of CLN, has been shown to be vectored by corn rootworm beetles.
Symptoms of the infection in a plant of either MCMV or MDMV.sub.B include mottling of the leaves, in a light greenish color. When both of the viruses occur in the same plant, i.e. CLN, there is a bright yellow-green mottling of the leaves which persists to the end of the growing season. CLN can infect plants at all stages of development, and the yield loss is greatest when the infection occurs at the younger stages of the plant life. Toward the end of the season the leaves may die inwardly from the margins with eventual death of the mature plant usually from the top down. The ears of infected CLN plants often are small and distorted having very little kernel development. Some infected plants are barren if the infection occurred early in development.
Hitherto, few, if any, agronomically desirable varieties of corn having resistance to CLN and specifically to MDMV.sub.B and having the necessary agronomic traits for commercial production have been produced. Some resistant sources such as the material used as a donor Pa405 are known but the genetic background of Pa405 evidences agronomically undesirable characteristics such that Pa405 is not presently commercially viable in hybrid combinations. Given that pursuant to this invention it has been discovered that a number of genes control resistance to MDMV.sub.B, a progeny plant containing the desired mix of agronomic traits (which make it commercially viable under the industry standards of 1994) and CLN and specifically MDMV.sub.B resistant genes within its genome is expected to be an extremely rare occurrence indeed.
One of the fundamental principles of maize breeding is the production of a hybrid by the combination of two inbreds. Thus there is an ongoing development of new inbreds. An inbred is a plant which has become homozygous in almost all gene loci. There are two germplasm sources for producing new inbreds. One source is germplasm that has been genetically engineered; the second source is adapted germplasm. This invention relates to the use of adapted germplasm and not to genetically engineered germplasm. In a conventional breeding program, pedigree breeding and recurrent selection breeding methods are employed to develop new inbred lines with desired resistant traits. Maize breeding programs attempt to develop these inbred lines by self pollinating the plants and selecting the desirable plants from the populations. An inbred produces a uniform population of hybrid plants when crossed with a second, different homozygous line, i.e., inbred. Inbreds tend to have poor vigor and low yield; however, the progeny of an inbred cross usually evidences high vigor and high yield. The progeny of a cross between two inbreds is often identified as an F.sub.1 hybrid. The resultant F.sub.1 hybrids which may be heterozygous at a number of gene loci, are evaluated to determine whether they show the resistant trait and agronomically important and desirable traits. Identification of desirable agronomic traits has typically been done by breeders' expertise. A plant breeder identifies a desired trait for the area in which plants are to be grown and attempts to develop inbreds which appear to pass the desirable trait or traits onto the hybrid.
Conventional plant breeders rely on phenotypic traits of the inbreds for selection purposes. Modern plant breeding technology utilizes molecular techniques which allows selection at the chromosomal level. One method of looking at plant genotypes is to use Restriction Fragment Length Polymorphisms (RFLPs) which provide a more precise method for identifying the chromosomal regions which affect the agronomic traits in the plant genome which the plant breeder is attempting to introgress into the inbred line for ultimate expression in the hybrid.
RFLPs can be used to identify specific chromosomal regions. Maize has ten chromosomes. Each chromosome has a short arm with a distal and proximal end and a long arm having a distal and proximal end. Between the short arm proximal end and long arm proximal end is a centromere. Each chromosome is made up of strands of the deoxyribonucleic acid (DNA) molecule which have a specific nucleic acid sequence. Selected restriction endonucleases will identify a specific base sequence and cleave the DNA molecule wherever this sequence occurs. The resultant cleaved portions are called restriction fragments. These restriction fragments can be separated by size by electrophoresis through agarose gels.
The DNA of two individual maize plants will differ in sequence at a variety of sites. Because of this difference, restrictive endonucleases may cleave an individual's DNA at a different site or location than the other individual's DNA. A polymorphism in the length of restriction fragments is produced when the fragments of the two individuals have different lengths. A polymorphism is detected by placing the fragments on a agarose gel electrophoresis and allowing them to separate by size over distance. A southern blot is then used. The fragments of the DNA are physically transferred on to a membrane, then nucleic acid hybridization detects the sequences by hybridization of the single strand of DNA (probe) on the southern blot. The nucleic acid reforms double stranded DNA. A labelled probe is used to detect a particular (DNA) sequence. One method is to use a radioactive labelled probe such that the DNA fragment will be identifiable through autoradiography techniques.
A variety of genes have been mapped and identified using RFLPs. Certain polymorphisms (molecular markers) are used to identify chromosomal areas associated with certain traits. A large number of molecular markers including RFLPs have been applied to the maize genome and a detailed maize genetic linkage map that can be used to localize important genes has been constructed.
A variety of traits have been identified by RFLPs; for example, P1 pericarp color has been linked to UMC185(P1) on the short arm of chromosome one of the maize plant. Probes BNL6.29 and UMC85 on chromosome six of the maize plant have been identified with MDMV.sub.A resistance in maize. Likewise, a variety of other traits have been genetically identified and placed on the maize genetic linkage map.
It would appear that once a desired trait is recognized by virtue of having a desired gene located between flanking probes in a maize plant by the use of RFLPs, that trait should be readily introgressed into an inbred line. Unfortunately, although RFLPs are a tool which can be employed to help identify the chromosomal region to which the trait appears to be linked, RFLPs are not a solution in and of themselves, they are simply a tool of identification. Furthermore, it is difficult in many instances to identify polymorphisms for the cross making the location of the desired gene difficult to identify and/or select. It should be noted that the chromosomal regions associated with CLN and specifically MDMV.sub.B resistance have not been identified or mapped by probes.
Because of the difficulties attendant with working with the CLN specifically MDMV.sub.B trait almost nothing has hitherto been conclusively known about the genes responsible, for example the number of genes involved, their action, and where they are located on the maize chromosomes. There is a need for the identification of the location of genes associated with CLN and MDMV.sub.B which permit their tracking when introgressed into a new plant through traditional breeding techniques. There also remains a need for a method of transferring CLN and/or MDMV.sub.B resistance to a corn plant that has desirable agronomic traits. There remains a need for the development of CLN and MDMV.sub.B resistant inbreds and commercial viable hybrids.